In this proposal, the effect of ethanol-induced reduction in hepatocellular S-adenosylmethionine (SAM):Sadenosylhomocysteine (SAH) ratios on an important methyltransferase, guanidinoacetate methyltransferase (GAMT) will be examined. This methyltransferase catalyzes the transfer of a methyl group from SAM to guanidinoacetate (GAA) to form creatine. It has been recently established that GAA is primarily synthesized in the kidney and transported through the circulation to the liver where it is methylated to form creatine. Liver is the principa source of circulating levels of creatine for uptake by creatine-requiring tissues that have a hig, but variable energy demand such as skeletal and cardiac muscles Therefore, the impact of compromised GAMT function following ethanol exposure can lead to two distinct detrimental consequences: 1) It can decrease creatine availability for extra-hepatic tissues, including skeletal and cardiac muscles that rely on an intact hepatic creatine biosynthetic pathway for optimal functioning; and 2) it can cause liver injury due to the accumulation of the toxic creatine precursor, GAA. Based on these considerations, the following hypothesis has been formulated: Ethanol induced altered SAM:SAH impairs hepatic creatine synthesis via its effect on GAMT activity causing detrimental consequences in the liver as well as extra-hepatic tissues such as the skeletal and cardiac muscles. Furthermore, betaine supplementation by virtue of correcting the altered SAM:SAH ratio could normalize hepatic creatine biosynthesis and thereby prevent these detrimental effects. The specific aims to test this hypothesis are: 1) To further examine and delineate the mechanism of impaired hepatic creatine biosynthesis by chronic ethanol administration. 2) To determine the effects of ethanol and betaine on circulating creatine and GAA levels, liver creatine efflux and kidney GAA production. 3) To demonstrate the cytotoxic consequences of ethanol-induced hepatic accumulation of creatine precursor, GAA. 4) To evaluate the effects and functional consequences of altered creatine biosynthesis and availability following ethanol exposure on skeletal and cardiac muscles and to examine whether betaine and/or creatine supplementation can revert or prevent the alcohol-induced changes. The objectives of this proposal are to first determine the role of ethanol-induced reduced liver SAM: SAH ratios in regulating hepatic creatine synthesis. Then, whether betaine by correcting the SAM: SAH ratio also reverses alcohol-induced effects on the liver creatine biosynthetic pathway will be examined. Since the body's requirement for creatine is met via uptake from the circulation, the effect of ethanol and betaine treatment on plasma levels of creatine and GAA will be determined next. Further, whether kidney's ability to produce and release GAA is altered by ethanol and/or betaine will be examined. In the next specific aim the detrimental consequences of accumulation of creatine precursor, GAA in the liver will be determined. Finally, the biochemical and functional consequences of creatine shortage in the skeletal and cardiac muscles of rats consuming ethanol will be examined to determine whether these detrimental consequences could be reversed by supplementing betaine and/or creatine in the diet. Overall, completion of these studies in an ethanol model of liver injury and validated using a GAMT knockout mouse model will provide insight in to the importance of maintaining the essential methylation reaction catalyzed by GAMT in the liver in relation to hepatic and extra hepatic tissues. PUBLIC HEALTH RELEVANCE: Chronic alcoholism and alcohol-induced liver disease and its associated multi-organ involvement are common factors associated with the hospitalization of our veterans. The objectives of this proposal are to determine the mechanism of impaired hepatic creatine synthesis following ethanol exposure as well as to examine the functional consequences of this defect in hepatic and extra-hepatic tissues such as the cardiac and skeletal muscles. Further this project also seeks to determine whether these deleterious effects can be reversed by supplementing betaine or creatine in the diet or would require a combined treatment modality. This proposal is clinically relevant and complements the commitment of VA Research to acquire knowledge of the impact of alcohol on liver and other organs in hopes of developing a therapeutic intervention of using betaine and/or creatine to manage alcoholic liver injury as well as associated myopathies prevalent among veterans.